The determination of the amount of phosphorous in bodily fluids such as serum or urine is important since determination of a build-up of phosphorous in bodily fluids can allow for the diagnosis of diseases such as uremia and chronic renal diseases where phosphorous retention occurs.
Quantitative determination of phosphorous in bodily fluids has been conventionally performed photometrically by use of the molybdenum blue reaction (I.M.Kolthoff and P.D. Elving. editors. part II, Volume V pages 317-402 1961). This reaction involves the formation of a phosphate molybdate complex which is then reduced by means of a reducing agent selected from the group consisting of phenylhydrazine, ascorbic acid, amino naphthol sulfonic acid or other reducing agents. Upon reduction, a blue colored complex of heteropolyacid is formed and absorbance of this complex is measured at around 700 nanometers.
Several disadvantages are associated with this conventional method for quantitatively determining the phosphorous in bodily fluids. First, it is necessary to have a protein free serum sample in order to perform this test, and preparation of such a protein free serum sample is cumbersome. Secondly, the sensitivity of this test is low. Finally, in order to perform this test, two sequential additions of reagent are required. That is, first the phosphate molybdate complex is formed, and then as noted above it is reduced. Such a two step process reduces the speed with which quantitative tests for phosphorous can be performed. Since the emphasis is for the quick and accurate completion of many tests, this latter disadvantage is particularly serious.
An attempt to overcome this latter disadvantage is disclosed in U.S. Pat. No. 3,795,484 which discloses a phosphorous determination which involves measurement of the unreduced phosphomolybdate complex at around 340 nanometers.
More specifically, this process of U.S. Pat. No. 3,795,484 comprises the steps of
(a) forming a mixture of the phosphate containing fluid and an ammonium molybdate solution;
(b) measuring by means of a centrifugal analytical photometer, a first absorbance reading at 340 nanometers within two seconds after said mixture is formed;
(c) measuring a second absorbance reading at 340 nanometers within ten minutes after said mixture is formed;
(d) comparing the absorbance differential with at least one other differential obtained simultaneously under the same conditions from a fluid containing a known concentration of phosphorous, and
(e) determining the amount of phosphorous in the phosphate containing fluid.
Several disadvantages are associated with this process. First, the process requires that the first absorbance reading must be obtained within two seconds of mixing the reagent with the sample of bodily fluid. Such a limitation makes this prior art process cumbersome to perform. Second, because there exists a difference between the matrices of protein (e.g., serum) and aqueous-based samples that causes the protein-based samples to react faster than aqueous samples, this prior art process requires a separate calibration for a phosphorous determination on an aqueous sample of bodily fluid such as urine and a protein sample of bodily fluid such as serum.
It is an object of the present invention to provide a process for the quantitative determination of phosphorous in bodily samples which involves a photometrically measured molybdate forming reaction which proceeds slowly enough so that the first of two absorbance readings may be taken as late as ten seconds after reagent is mixed with the bodily fluid to be tested.
It is another object of the present invention to provide a process for the quantitative determination of phosphorous in bodily fluids such that the same calibration can be used whether an aqueous bodily fluid such as urine or a protein-based bodily fluid such as serum is tested. By providing such a process, one would be able to run aqueous and serum samples for a quantitative determination of the phosphorous simultaneously.